WO2007125822A1 - Complexe utilisable pour le criblage d'une substance - Google Patents

Complexe utilisable pour le criblage d'une substance Download PDF

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Publication number
WO2007125822A1
WO2007125822A1 PCT/JP2007/058582 JP2007058582W WO2007125822A1 WO 2007125822 A1 WO2007125822 A1 WO 2007125822A1 JP 2007058582 W JP2007058582 W JP 2007058582W WO 2007125822 A1 WO2007125822 A1 WO 2007125822A1
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WIPO (PCT)
Prior art keywords
protein
substance
carrier
physiologically active
active substance
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PCT/JP2007/058582
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English (en)
Japanese (ja)
Inventor
Hiroshi Handa
Chika Sawa
Yasuaki Kabe
Satoshi Sakamoto
Kousuke Nishio
Tetsu Mau Chit Yung
Chikanori Kuramori
Shoichiro Furukawa
Satoshi Inoue
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Tokyo Institute Of Technology
Chisso Corporation
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Application filed by Tokyo Institute Of Technology, Chisso Corporation filed Critical Tokyo Institute Of Technology
Priority to JP2008513170A priority Critical patent/JPWO2007125822A1/ja
Publication of WO2007125822A1 publication Critical patent/WO2007125822A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention relates to a complex effective for screening of a physiologically active substance, and a drug screening method using the same.
  • Patent Document 1 a magnetic bead in which ferrite nanoparticles are coated with an organic polymer. Since these magnetic beads can be separated and recovered by magnetic force, they are expected to enable high-speed, automatic screening of proteins and the like.
  • Patent Document 1 JP-A-2006-88131
  • the present invention provides a complex effective for screening a physiologically active substance, and a method for screening a drug using the same.
  • it acts on a protein that causes disease, but has strong side effects. Therefore, it effectively uses a large number of compounds that cannot be used as pharmaceuticals and derivatives of the compounds, and is a drug candidate. It becomes possible to efficiently search for a compound that becomes By conducting screening using the complex of the present invention, it becomes possible to efficiently develop new drugs and the like.
  • the inventors of the present invention have made extensive studies in order to solve the above problems. As a result, if the target protein is fixed to the carrier via a physiologically active substance that binds to the protein, and there is another physiologically active substance that binds to the target protein, Paying attention to the competitive release of the protein in the carrier, labeling the target protein with a luminescent substance (luminescent substance and fluorescent substance), etc., thereby releasing the target protein (i.e., another protein that binds to the protein). Based on this finding, the present inventors have completed the present invention.
  • the present invention provides the following (1) to (: 16).
  • a complex comprising a carrier, a physiologically active substance, and a protein, wherein the carrier is bound to the physiologically active substance, and the physiologically active substance has an affinity for the protein, depending on the affinity.
  • a method for screening a drug characterized by using the complex according to any one of (1) to (7).
  • (9) a step of coexisting a test compound and the complex described in (1) to (7) above or in any case in a solvent, a step of separating the complex from the solvent, and a luminescence intensity of the solvent Or the method for screening a drug according to (8), further comprising a step of measuring fluorescence intensity.
  • (10) A step in which at least a protein labeled with a luminescent substance or a fluorescent substance, a carrier bound to a physiologically active substance having an affinity for the protein, and a test compound coexist in a solvent.
  • a method for screening a drug comprising a step of separating a carrier from the solvent and a step of measuring the emission intensity or fluorescence intensity of the solvent.
  • At least a protein labeled with a luminescent substance or a fluorescent substance, a physiologically active substance having affinity for the protein and a quenching carrier, and a test compound coexist in a solvent.
  • a method for screening a drug having a step of measuring the emission intensity or fluorescence intensity of the solvent.
  • the physiologically active substance having an affinity for the protein is different from the physiologically active substance used in the complex.
  • a physiologically active substance that bind to disease-causing proteins but cannot be used as pharmaceuticals due to side effects.
  • FIG. 1 is a diagram schematically showing a first screening method of the present invention.
  • A shows the case where the test substance binds to the protein
  • B shows the case where the test substance does not bind to the protein.
  • FIG. 2 is a diagram schematically showing the second screening method of the present invention.
  • A shows the case where the test substance binds to the protein
  • B shows the case where the test substance does not bind to the protein.
  • FIG. 3 shows an example of a screening method using a magnetic carrier (1).
  • a carrier-bioactive substance-protein complex
  • b screening by competitive action and release of protein by competitive inhibition.
  • FIG. 4 shows an example of a screening method using a magnetic carrier (2).
  • c carrier-bioactive substance-protein complex
  • d screening by competitive action and release of protein by competitive inhibition
  • e detection of binding between test compound and protein.
  • FIG. 5 is a diagram showing the results of an experiment conducted in Reference Example 2.
  • FIG. 6 shows the results of the experiment performed in Reference Example 3.
  • FIG. 7 is a diagram showing the results of an experiment conducted in Example 1. * Indicates rHis-DHFR-AQ, * * indicates r His-DHFR-AQ degradation product.
  • FIG. 8 shows the results of the experiment (protein detection) performed in Example 3.
  • MTX represents methotrexate
  • SRM represents streptomycin
  • PNC penicillin
  • FIG. 9 is a diagram showing the results of an experiment (luminescence intensity measurement) performed in Example 3.
  • MTX represents methotrexate
  • SRM represents streptomycin
  • PNC penicillin
  • the complex of the present invention includes a carrier, a physiologically active substance, and a protein.
  • the carrier is bound to the physiologically active substance, and the physiologically active substance has an affinity for the protein, and depending on the affinity. It is bound to a protein, and the protein is labeled with a luminescent substance or a fluorescent substance.
  • the form of the bond between the carrier and the physiologically active substance is not particularly limited, but it is preferably a relatively strong bond that does not dissociate when the complex of the present invention is used for screening.
  • the binding between the physiologically active substance and the protein is not particularly limited as long as it can be bound by the affinity of both, but has an affinity for the protein when the complex of the present invention is used for screening. It is preferably a relatively weak bond that dissociates when another physiologically active substance is present. Examples of such binding include binding between a receptor protein and a ligand, binding between an enzyme protein and its substrate, and the like.
  • the carrier is usually in the form of particles, but is not particularly limited as long as it can bind to a physiologically active substance.
  • a carrier such as a substrate may be used.
  • the carrier is preferably one having a property that can be easily separated from the solvent, for example, a magnetic carrier.
  • the magnetic carrier include ferrite particles having an organic polymer coating developed by the present inventors. Ferrite particles having an organic polymer coating can be produced, for example, as follows.
  • the surface of a ferrite particle synthesized by an aqueous solution process is formed with a surfactant double membrane. Cover it. By covering the surface with surfactant micelles, it is possible to facilitate the introduction of the hydrophobic polymer on the surface of the ferrite particles. It is possible to coat the surface of the ferrite particle with a polymer by emulsion polymerization of the bull monomer in the presence of the ferrite particle coated with the double film.
  • the particle diameter of the ferrite particles having an organic polymer coating is not particularly limited, but is preferably in the range of 5-300 nm.
  • the organic polymer to be coated include a copolymer with a styrene monomer based on styrene, or a hydrophilic polymer such as polyacrylic acid or polybulal alcohol.
  • PEG, dextran, phospholipid, nucleic acid, protein, etc. may be used as the coating material.
  • a carrier having a quenching property may be used as the carrier.
  • a screening method (FIGS. 1 and 2) without a step of separating the released protein and the complex can be performed.
  • the quenching carrier include ferrite particles having the above-mentioned organic polymer coating, but particles having a quencher substance bonded thereto or the quencher substance itself may be used.
  • Quenchia substances include Dabcyl, BHQ1, BHQ2, and Eclipse TM Dark Quencher.
  • a quencher substance may be bonded to the ferrite particles having an organic polymer coating to further enhance the quenching property.
  • the protein is not particularly limited, and examples thereof include a receptor for a physiologically active substance and an enzyme using the physiologically active substance as a substrate. These are preferably used when the physiologically active substance is a drug for diseases.
  • the protein is preferably a protein that causes disease. Examples of such proteins include dihydrofolate reductase (DHFR) as an enzyme, amyloid (A j3) as an amyloid protein, and pael receptor (Pael Rec mark tor) as a receptor.
  • DHFR dihydrofolate reductase
  • a j3 amyloid
  • pael receptor Pael Rec mark tor
  • physiologically active substance to be used is not particularly limited, when the use of the complex of the present invention is a drug screening method, it binds to a protein causing a disease.
  • the fluorescent substance used for labeling is not particularly limited, and uses a low-molecular organic compound having fluorescence ability such as FITC, TRITC, TAMRA, Cy3, Cy5, green fluorescent protein (GFP) which is a fluorescent protein having fluorescence ability, etc. be able to.
  • a low-molecular organic compound having fluorescence ability such as FITC, TRITC, TAMRA, Cy3, Cy5, green fluorescent protein (GFP) which is a fluorescent protein having fluorescence ability, etc. be able to.
  • the luminescent substance used for labeling is not particularly limited, and a luminescent organism-derived calcium-binding luminescent protein, luciferase, or other luminescent protein can be used. Furthermore, a protein that catalyzes a chemical luminescence reaction system using alkaline phosphatase or horseradish peroxidase can also be used as a luminescent substance.
  • aequorin which is one of the most sensitive and non-toxic calcium-binding photoproteins.
  • Aequorin is a calcium-binding photoprotein that emits light instantaneously upon contact with trace amounts of calcium ions (10-ioles / liter or more). Luminous sensitivity is also very high with a detection limit of S0.1 picogram or less in a commercially available detector.
  • the complex of the present invention can be used in a drug screening method.
  • the specific method is not particularly limited, but the step of allowing the test compound and the complex of the present invention to coexist in a solvent, the step of separating the complex of the present invention from this solvent, and the emission intensity of the solvent or A method having a step of measuring fluorescence intensity can be given as an example (hereinafter, this screening method may be referred to as “first method”).
  • the solvent used in the first method is not particularly limited, and examples thereof include water containing an appropriate buffer.
  • the coexistence time is not particularly limited, but is usually about 1 hour.
  • the method for separating the complex of the present invention from the solvent is not particularly limited, and the carrier may be collected at a specific location in the solvent from which the carrier may be removed.
  • the carrier is magnetic, for example, as described later, a magnet is placed around the well containing the solvent, the carrier is moved to the peripheral part of the well, and the cylindrical partition is placed in the well. Can be shown.
  • At least a protein labeled with a luminescent substance or a fluorescent substance as a method for screening a drug utilizing the competitive action between a test compound and a physiologically active substance, and the protein A carrier bound to a physiologically active substance having an affinity for the compound, and a test compound And a step of separating the carrier from the solvent, and a method of measuring the emission intensity or fluorescence intensity of the solvent (hereinafter, this screening method is referred to as “second method”). It may be called "method”.)
  • the carrier, physiologically active substance, protein, fluorescent substance, luminescent substance and the like to be used may be the same as in the first method.
  • a physiologically active substance that interacts with a specific protein can be obtained.
  • a physiologically active substance can be a candidate substance for a novel drug.
  • FIG. 3 An example of a screening method using a magnetic carrier as the first method will be described with reference to FIGS. 3 and 4.
  • FIG. 3 An example of a screening method using a magnetic carrier as the first method will be described with reference to FIGS. 3 and 4.
  • FIG. 3 An example of a screening method using a magnetic carrier as the first method will be described with reference to FIGS. 3 and 4.
  • FIG. 3 An example of a screening method using a magnetic carrier as the first method will be described with reference to FIGS. 3 and 4.
  • a complex comprising a magnetic carrier [6], a physiologically active substance [7], and a target protein [8] labeled with the photoprotein aequorin [9] is divided into 96 well plates [11].
  • Note Figure 3A. Add chemical library or fungus culture library [10] (substances contained in these libraries are the test compounds) to the wells ( Figure 3B). Place 96-well plate [11] on the stirring table and allow competitive inhibition reaction for 30 minutes to 1 hour. Place a ring-shaped magnet [12] plate that fits the size of the well under the 96-well plate [11] ( Figure 3C).
  • the magnetic support [6] gathers outside the well, and the released aequorin-labeled protein [8] does not move outward (FIGS. 3D and 4E).
  • a portion of the aequorin-labeled protein [8] released here is collected, CaCl solution is injected, and the luminescence intensity is measured with a plate reader.
  • a cylinder [13] with a diameter smaller than that of the ring magnet [12] is fitted onto the 96 well plate [11] that has undergone competitive inhibition (Fig. 4F).
  • the carrier [6] having magnetism and the released target protein [8] can be separated.
  • the CaCl solution is injected into the cylinder [13] and the luminescence intensity is measured with a plate reader (Fig. 4G).
  • the magnetic carrier [6] can be gathered outside, and by inserting the cylinder [13], the carrier [6] It is possible to separate the released protein [8]. By separating the carrier [6] and the released protein [8] in this way, only the released protein [8] is reacted with the CaCl solution and attached to the carrier [6].
  • the bioactive substance [2] is bound to the quenching carrier [1].
  • this carrier [1] is labeled with a luminescent substance or fluorescent substance [4]
  • the protein [3] and the test compound [5] coexist, the test compound [5] becomes the protein [3]. ]
  • the protein [3] binds to the test compound [5] as well as the physiologically active substance [2].
  • the protein [3] increases. For this reason, the amount of luminescent materials (white stars in the figure) that are not quenched by the carrier increases, and the luminescence intensity in the solvent is maintained in a strong state.
  • the test compound [5] does not bind to the protein [3]
  • the protein [3] binds only to the physiologically active substance [2], so that the number immobilized on the carrier [1] increases. Conversely, free protein [3] is reduced. For this reason, the number of luminescent substances (black stars in the figure) quenched by the carrier increases, and the luminescence intensity in the solvent becomes weaker.
  • the protein [3] labeled with a luminescent or fluorescent substance [4] is immobilized via a quenching carrier [1] and a physiologically active substance [2].
  • the carrier [1] is allowed to coexist with the test compound [5]
  • the test compound [5] binds to the protein [3]
  • a competitive action occurs.
  • the protein [3] is released from the carrier [1].
  • Luminescent or fluorescent materials [4] shine strongly when not in close proximity to the quenching carrier [1] (white stars in the figure), but light is weak when in close proximity to the quenching carrier [1]. (Black star in the figure). Therefore, the protein [3] is released and emits strong light.
  • the test compound [5] does not bind to the protein [3]
  • the protein [3] remains immobilized on the carrier [1], and the light emitted from the phosphor or fluorescent substance [4] is weak. Remain.
  • ferrite particles having an organic polymer coating were prepared as follows.
  • Hydrophobic ferrite particles were obtained by saturated adsorption of undecenoic acid to 150 mg of ferrite particles with an average particle size of 40 nm, which were precipitated in aqueous solution.
  • Emulgen 1 150S-70 manufactured by Kao Corporation having a PE 0 chain was sonicated into this hydrophobized phosphor particle, and this nonionic surfactant was prepared by sonication. Surfactant was adsorbed on the surface of the hydrophobized ferrite particles to rehydrophilize the particles and obtain a colloidal solution of rehydrophilized ferrite particles dispersed in water.
  • a monomer mix having 2.7 g of styrene, 0.3 g of GMA (glycidyl methacrylate) and 0.08 g of DVB (dibutylbenzene, cross-linking agent) is added to the colloid solution so that the total amount becomes 125 g.
  • GMA glycidyl methacrylate
  • DVB dibutylbenzene, cross-linking agent
  • the emulsion polymer particles thus obtained were washed to obtain ferrite particles (hereinafter sometimes referred to as “magnetic beads”) having an organic polymer film.
  • ferrite particles hereinafter sometimes referred to as “magnetic beads”.
  • the particles were monodisperse particles having an average particle diameter of 200 nm and had ferrite particles inside.
  • the supernatant was removed by centrifugation at 0 mM 40 ul 0 l 760 ul 800 ul, and 200 ⁇ of 100 mM H-signed es_NaOH was added to redisperse the magnetic beads. This operation was repeated 8 times to wash the magnetic beads.
  • FITC-immobilized magnetic bead suspension (0.4 mg / 200 ⁇ ) 50 ⁇ 1 and 0.1 M DTT solution 50 ⁇ 1 were mixed, and fluorescence intensity was measured 45 minutes later.
  • the fluorescence intensity was measured using a magnetic bead suspension (DTT +), a solution after removing magnetic beads from the suspension (after magnetic separation (DTT +)), and a magnetic bead suspension that did not contain a 0.1M DTT solution.
  • the test was performed for three types of suspension (DTT—). The results are shown in Fig. 5.
  • the fluorescence intensity increased by releasing FITC from the magnetic beads by DTT, and the fluorescence intensity further increased by removing the magnetic beads in the liquid.
  • dabsyl acid 50 mg was dissolved in 1500 ⁇ l of DMF to prepare a 66.7 mg / ml dabsylate solution.
  • the FITC-immobilized magnetic beads prepared in Reference Example 2 were suspended in 1000 ⁇ l of DMF to prepare a 1000 ⁇ m FITC-immobilized magnetic bead suspension. This suspension, dabsinore solution and DMF were mixed to prepare a magnetic bead suspension having the composition shown in Table 2.
  • the release of FITC from the magnetic beads by DTT increases the fluorescence intensity, but the rate of increase largely depends on the amount of modified dabsyl.
  • the fluorescence intensity after DTT treatment was about 15 times the fluorescence intensity without DTT treatment. (Bar 5 in the figure) was about 30 times.
  • This low temperature induction system is commercially available from Takara Shuzo. It is expressed by the E. coli cold shock gene cspA promoter contained in the expression vector pColdll. A lac operator is inserted downstream of the cspA promoter to strictly control expression.
  • E. coli expressing rHis-DHFR-apoAQ was recovered by centrifugation and washed with PBS. colon The bacteria were suspended in 50 ml of a lysis solution (20 mM Tris_HCl (pH 7.9), 300 mM NaCl, lOmM Imidazole, 0.1% NP-40), and Escherichia coli and the genome were disrupted by ultrasound. The insoluble fraction was separated by centrifugation, and the soluble fraction was mixed with Ni-NTA Agarose (QIAGEN). Four. Rotated for 1 hour at C to bind rHis-DHFR-apoAQ to nickel agarose gel.
  • elution solution (20 mM Tris-HCl (pH 7.9), 300 mM NaCl, lOOmM Imidazole) was added to the column to elute rHis-DHFR-apoAQ from a nickel agarose gel.
  • the elution fraction was collected by dispensing 500 ⁇ l each. rHis-DHFR-apoAQ was recovered in the second fraction.
  • Regeneration reaction solution 500 ⁇ 1 rHis-DHFR-apoAQ, 10 ⁇ 1 0.5 ⁇ EDTA ⁇ 2 ⁇ 1 2_mercaptoethanol, 40 ⁇ g serentrazine, 4.5 ml TEdOmM Tris-HCl (pH 7.9), lOmM EDTA) Placed in a 15 ml tube and rotated at 4 ° C for 3 hours. Every 30 minutes, the reaction tube was opened and oxygen was supplied.
  • Serentrazine is not a commercially available method (Inoue, S., Su giura, S., Kakoi, H., Hasizume, K., Goto, T., and no, H. (197 ⁇ Chem Lett. ⁇ 41— 14 The product synthesized according to 4) was used, rotated at 4 ° C for 3 hours, and then allowed to stand overnight at 4 ° C.
  • 10 ml TE (10 mM Tris-HCl (pH 7.9), lOmM EDTA), 10 ml wash TEl (10 mM Tris-HCl (pH 7.9), lOmM EDTA, 50 mM NaCl), 10 ml wash TE2 (1 OmM Tris-HCl (pH 7) .9), lOmM EDTA, lOOmM NaCl) were slowly and slowly added to wash the column.
  • 10 ml elution TE (10 mM Tris-HCl (pH 7.9), lOmM EDTA, 400 mM NaCl) was slowly added gently to elute rHis_DHFR-AQ. The elution fraction was collected by dispensing 500 ⁇ l each. Proteins contained in the eluted fraction and the fraction collected during washing were separated by SDS-PAGE and stained with Coomassie brilliant blue. The result is shown in FIG.
  • rHis_DHFR-AQ was recovered in the 4th-5th fraction. Purified rHis-DHFR-AQ is dispensed at 100 z 1 into 1.5 ml Eppendorf tubes. Saved with C.
  • the magnetic beads were washed once with DMF, and then suspended in 430 ⁇ 1 of DMF.
  • 50 / il triethylamine and 20 ⁇ 20 acetic anhydride were added, and incubated at room temperature for 2 hours. After washing once with water, it was suspended in 450 / il water.
  • 50 ⁇ l of IN NaOH was added Q, incubated at room temperature for 30 minutes, and then the magnetic beads were washed 5 times with water, suspended in 500 ⁇ l of water and stored.
  • the supernatant was removed by centrifuging the suspension, 1M ethanolamine DMF solution was added, and the mixture was allowed to stand at room temperature for 2 hours. Again, the supernatant was removed by centrifugation, and the magnetic beads were collected. The magnetic beads were washed three times with a 50% aqueous methanol solution and then stored in a 50% aqueous methanol solution.
  • Binding Buffer (10 mM HEPES, 150 mM KC1, 5 mM EDTA, 10% Glycerol, 0 ⁇ 1% ⁇ 40) 3 times Then, it was mixed with rHis-DHFR-AQ (lOOng / 100 ⁇ 1), reacted at 4 ° C. for 3 hours, and bound.
  • the rHis-DH is only mixed with MTX in the supernatant after the binding reaction. A band indicating FR-AQ was detected, and such a band was not detected when mixed with streptomycin (SRM) and penicillin (PNC).
  • SRM streptomycin
  • PNC penicillin
  • the luminescence intensity was measured by adding 50 ⁇ m of the solution.
  • the result is shown in FIG.
  • (-), (+), (++), and (++) indicate MTX by ⁇ fix solution A, MTX fix solution ⁇ , ⁇ X fix solution C and MTX fix solution D, respectively. Is used.
  • SRM streptomycin
  • PNC penicillin
  • FIG. 9 when mixed with streptomycin (SRM) and penicillin (PNC), almost no light was emitted, whereas when mixed with MTX, strong light was confirmed. Strong luminescence when mixed with MTX is thought to be due to the release of quenching magnetic bead force rHis-DHFR-A Q force S. This result is consistent with the result of electrophoresis described above.

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Abstract

La présente invention concerne un complexe comprenant un support, une substance physiologiquement active et une protéine, le support se liant à la substance physiologiquement active, la substance physiologiquement active ayant une affinité pour la protéine qui lui permet de se lier à elle, et la protéine étant marquée avec une substance luminescente ou fluorescente. L'invention concerne également un procédé de criblage d'une substance à l'aide du complexe. Le procédé permet d'utiliser avantageusement un composé qui peut agir sur une protéine pathogène mais qui ne peut pas être utilisé en tant qu'agent pharmaceutique en raison de graves effets secondaires défavorables et il permet également de rechercher une substance avec une efficacité élevée.
PCT/JP2007/058582 2006-04-28 2007-04-20 Complexe utilisable pour le criblage d'une substance WO2007125822A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011021581A1 (fr) * 2009-08-20 2011-02-24 学校法人早稲田大学 Marqueur sélectif pour biopolymère cible

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JPH11155598A (ja) * 1997-09-23 1999-06-15 Becton Dickinson & Co 蛍光消光による核酸の検出
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JP2002090366A (ja) * 2000-09-14 2002-03-27 Rikogaku Shinkokai フェライトを固定した生体特異的親和性キャリヤとその製造方法
JP2006505775A (ja) * 2002-11-07 2006-02-16 エラスムス　ユニフェルシテイト　ロッテルダム 相互作用する分子を検出するためのｆｒｅｔプローブおよび方法

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Publication number Priority date Publication date Assignee Title
JPH02195256A (ja) * 1989-01-24 1990-08-01 Matsushita Electric Ind Co Ltd 免疫的検出方法および装置
JPH0534346A (ja) * 1990-09-14 1993-02-09 Tosoh Corp 免疫測定法
JPH0782302A (ja) * 1993-07-20 1995-03-28 Nippon Paint Co Ltd ポリマーで保護被覆されたフェライト被覆磁性粒子及びその製造法
JP2001502055A (ja) * 1996-10-04 2001-02-13 ワラック オサケユイチア ホモジニアスルミネセンスエネルギー転移アッセイ
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